A variety of objects have been imaged using a variety of approaches, for acquiring a wealth of information. One imaging application involves the imaging of small or microscopic objects, using a microscope-type component. Various such approaches have involved the use of a different techniques for obtaining an image, such as by collecting light or other electromagnetic radiation.
However, obtaining information from small objects can be challenging. For instance, it can be difficult to image single molecules that can have fixed orientations of their emission dipoles, which can lead to asymmetric features in their fluorescence emission patterns. These anisotropic features can cause systematic mislocalization of the positions of individual molecules, which can ultimately lead to degraded resolution and imaging artifacts. This problem can be compounded by the effects of slight microscope defocus (e.g., |Δz|≦250 nm), which enhances asymmetric features and may lead to x-y errors (e.g., of up to ˜200 nm). In this context, when a source of light is positioned at the focal plane of a microscope, rays of light emanating from the source will be collected by the microscope's optics, and refocused onto a detector (such as a camera sensor). The image formed by a single point of light is called the microscope's point-spread-function (PSF). While a microscope's PSF is useful for imaging two-dimensional slices of a specimen, objects that are a small distance away from the microscope's focal plane may appear blurry. Furthermore, it can be challenging to precisely measure relative depths of objects, as related to their distance from a plane of focus.
These and other matters have presented challenges to imaging, for a variety of applications.